Europe PMC Funders Group Author Manuscript Sci Signal. Author manuscript; available in PMC 2013 January 09. Published in final edited form as: Sci Signal. ; 3(134): ra60. doi:10.1126/scisignal.2001104. Europe PMC Funders Author Manuscripts The PI3K Isoforms p110α and p110δ are Essential for Pre-B Cell Receptor Signaling and B Cell Development Faruk Ramadani1, Daniel J. Bolland2, Fabien Garcon1, Juliet L. Emery1, Bart Vanhaesebroeck3, Anne E. Corcoran2, and Klaus Okkenhaug1,* 1Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge CB22 3AT, UK 2Chromatin and Gene expression, The Babraham Institute, Cambridge CB22 3AT, UK 3Centre for Cell Signaling, Institute of Cancer, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK Abstract B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes are assembled in frame to produce a functional B cell receptor (BCR) and antibodies. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igα and Igβ chains. Whereas the activation of Src and Syk tyrosine kinases is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110δ isoform of phosphoinositide 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on tonic BCR signaling, are not substantially affected by a deficiency in p110δ. Here, we show that in the absence of p110δ, Europe PMC Funders Author Manuscripts p110α, but not p110β, can compensate to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110α and p110δ activities, pre-BCR signaling fails to suppress the production of recombination-activating gene (Rag) protein and to promote developmental progression of B cell progenitors. By contrast, p110α does not contribute to agonist-induced BCR signaling. These studies indicate that either p110α or p110δ can mediate tonic signaling from the BCR, but that only p110δ can contribute to antigen-dependent activation of B cells. Introduction B cell development occurs in the bone marrow, where the gradual acquisition of B cell characteristics correlates with the loss of potential for differentiation into other blood cell lineages (1). B cells are defined by the surface expression of the B cell receptor (BCR), which is encoded by rearranged immunoglobulin (Ig) heavy chain (Igh) and Ig light chain (Igκ or Igλ) genes. The Igh locus comprises multiple Variable (V), Diversity (D) and Joining (J) gene segments. First a D segment is joined to a J segment and then a V segment is joined to a DJ segment to form a VDJH recombined Igh gene. Before this can occur, the interleukin-7 receptor (IL-7R) stimulates chromatin changes in the Igh locus rendering it accessible for recombination activating gene (Rag1 and Rag2) proteins that catalyze VDJH *To whom correspondence should be addressed. [email protected]. Author contributions: FR, DJB, FG, JE and KO performed experiments, BV contributed essential research tools, AEC and KO supervised experiments, KO wrote the paper. All authors revised drafts of the paper. Competing interests: BV and KO act as scientific advisers to Intellikine and GlaxoSmithKline, respectively. Ramadani et al. Page 2 recombination (2). If the Igh gene segments are rearranged in-frame, then the Igμ heavy chain forms a pre-BCR in association with the surrogate light chains λ5 and VpreB on the cell surface. After several rounds of division, during which the Rag genes are temporarily turned off, the Igκ or Igλ locus, each of which comprises multiple V and J gene segments, is rearranged to form Igκ or Igλ genes. Igκ or Igκ light chain proteins replace the surrogate light chains to form the mature BCR with the Igμ heavy chain. B cell precursors that lack Europe PMC Funders Author Manuscripts Rag1, Rag2 or the transmembrane domain of Igμ (μMT) are blocked in their development at the pro-B cell stage (3-5). These observations demonstrate the existence of a developmental checkpoint that only permits pre B cells with in-frame rearranged Igμ heavy chains to develop further. There is increasing evidence that the pre-BCR transmits signals without being clustered by specific agonists (6). Pre-BCR signaling is initiated by the activation of Src family tyrosine kinases that phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) within the invariant Igα and Igβ transmembrane proteins that form a complex both with the pre-BCR and later with the BCR (6). The tyrosine kinase Syk is recruited to phosphorylated Igα and Igβ and it plays an important role in the development of immature B cells in the spleen (7). Together with the related tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP-70), Syk is essential for pre-BCR signaling (8). Src homology 2 (SH2) domain– containing leukocyte adaptor protein of 65 kD (SLP-65, also known as BLNK) is an adaptor protein that links Syk to the activation of phospholipase c γ (PLC-γ). SLP-65-deficient pre- B cells are partially blocked at pre-B cell stage of development; however, the pre-B cells continue to proliferate and eventually develop into pre-B tumor cells (9-11). These results implicate additional signals downstream of Syk that are also important for pre-BCR signaling. Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes that phosphorylate the 3- position of the phosphatidylinositol (PtdIns) ring. Class I PI3Ks use the substrate PtdIns-4,5- bisphosphate (PIP2) to generate PtdIns-3,4,5-trisphosphate (PIP3) (12, 13). PIP3 acts as a membrane tether for proteins such as Akt and Btk in B cells. Akt can stimulate the serine Europe PMC Funders Author Manuscripts and threonine kinase mammalian target of rapamycin (mTOR ) and suppress Foxo transcription factors, whereas Btk contributes to the activation of PLC-γ. Class I PI3Ks integrate a number of signaling events that are controlled by Syk, because key proteins that are phosphorylated by Syk, including CD19, B-cell adapter for phosphoinositide 3-kinase (BCAP), and the guanine nucleotide exchange factor Vav, contribute to the activation of PI3K as initiated by the pre-BCR or the BCR (14, 15). Syk may also directly regulate the activity of PI3K (16); however, the precise role of PI3K signaling, especially downstream of the pre-BCR is incompletely understood. Tyrosine kinases are linked to the activation of subset of PI3Ks (class IA), which are associated with p85 regulatory subunits that can bind to proteins that contain phosphorylated tyrosine residues. Mammals have three genes, Pik3r1, Pik3r2, and Pik3r3, which encode the class IA PI3K regulatory subunits p85α, p85β. and p55γ respectively. The subunits p55α and p50α are generated by alternative start codon usage from Pik3r1. Pik3ca, Pik3cb, and Pik3cd encode the class IA PI3K catalytic subunits p110α, p110β, and p110δ, respectively, each of which can bind to any of the regulatory subunits. Studies to date suggest a non-essential role for PI3K in early B cell development. Thus both p85α and p110δ-deficient mice showed near normal B cell development in the bone marrow (17-24). After development in the bone marrow B cells circulate through the blood and lymph and populate the spleen, lymph nodes and plural cavities where they continue their development. There are three main subtypes of mature B cell populations which are defined by their preferred anatomical location and cell surface phenotypes: follicular (FO) B cells, also known as B2 cells, are found in the follicles in the spleen and lymph nodes, marginal Sci Signal. Author manuscript; available in PMC 2013 January 09. Ramadani et al. Page 3 zone (MZ) B cells are found in the MZ at the perimeter of the follicles of the spleen, whereas B1 cells are found primarily in the pleural cavities such as the peritoneum. In both p85α-deficient and p110δ-deficient mice there were reduced numbers of follicular B cells, and peritoneal B1 and splenic MZ B cells were almost absent (17-24). Additional studies have shown that “tonic” PI3K signaling is required to suppress the expression of Rag and to promote the survival of immature and mature B cells (25-28). PI3K suppresses the Europe PMC Funders Author Manuscripts expression of Rag by virtue of its capacity to terminate transcription of Foxo which binds to the promoters of Rag1 and Rag2 (29, 30). Thus, a picture emerged in which p85α recruits p110δ to a BCR-associated signaling complex, and this interaction is essential for the development of B1 and MZ B cells, but is not essential for the development of B cell precursors in the bone marrow and follicular (FO) B cells in the spleen. Here, we report that similar to what occurs in p110δ-deficient mice, the development of B cells in the bone marrow occurred normally in mice with lymphocytes that lacked either p110α or p110β; however, the combined loss of p110α and p110δ resulted in a near complete block in B cell development at the pre-B cell stage. Single-cell analysis revealed that B cells blocked at this stage contained Rag and had rearranged their heavy chain genes. Moreover, p110α and p110δ doubly-deficient pre-B cells failed to inhibit the expression of Rag in Igμ+ cells. Consequently, we observed increased proportions of pre B cells with both loci rearranged and which contained Igμ and Rag. Thus, p110α, but not p110β, compensated for the lack of p110δ in pre-B cells to provide PI3K activity that was essential for developmental progression; however, antigen-dependent activation of mature B cells was strictly dependent on p110δ.